Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye

Exp. Eye Res. (1995) 61, 223-234

Regulation of O u t f l o w Rate and Resistance in the Perfused Anterior Segment of the Bovine Eye M I C H A E L W I E D E R H O L T a * , S I M O N E B I E L K A a, F R I E D E R I C E S C H W E I G 5 ELKE LOTJEN-DRECOLLb AND A L B R E C H T L E P P L E - W l E N H U E S a

aInstitut for Klinische Physiologie, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Germany, bAnatomisches Institut II, Universitat Erlangen-Nornberg, Erlangen, Germany (Received Columbia 12 October 1994 and accepted in revised form 21 March 1995) Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper We characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8"8 mmHg. A constant outflow of approximately 6-8/~I rain -1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min #l -a. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30 %. ECs0for carbachol was 3 x 10 -8 mol 1-1.Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10 -~ tool 1 1 reduced outflow by 15 %. Epinephrine at 10 -5 mol 1-1 reduced outflow, while epinephrine at 10 6 tool 1-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 × 10 -9 and 2 x 10 8 mol 1 1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow. © 1995 Academic Press Limited Key words: perfusion of aqueous outflow pathway; bovine trabecular meshwork ; muscarinic receptors; adrenergic receptors; endothelin receptors. 1. Introduction It has long been postulated that trabecular meshwork cells may be contractile and that pilocarpine could influence outflow facility directly (Barfiny, 1962). More recently, re-examination of the h u m a n and bovine chamber angle has presented evidence for contractile filaments in trabecular meshwork (Ringvold, 1978; Tripathi and Tripathi, 1980, 1984; Grierson et al., 1986; DeKater, Spurr-Michaud and Gipson, 1990; F1/igel, Tamm and L/itjen-Drecoll, 1991; F1/igel et al., 1992). However, the functional significance of these contractile properties of trabecular meshwork is unclear. We have already shown the excitability of cultured bovine and h u m a n trabecular meshwork cells with electrophysiological methods (Coroneo et al., 1991: Wiederholt, LeppleWienhues and Stahl, 1993; Lepple-Wienhues et al., 1994). By directly measuring the force of isolated trabecular meshwork strips under isometric conditions, the contractility of the meshwork from bovine eyes has been demonstrated (Lepple-Wienhues, Stahl and Wiederholt, 1991a; Lepple-Wienhues et al., 1991b; Wiederholt, Sturm and Lepple-Wienhues,

* For correspondence at: Institut f/Jr Klinische Physiologie, Universitfitsklinikum Benjamin Franklin, Freie Universit~t Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. 0 0 1 4 4 8 3 5 / 9 5 / 0 8 0 2 2 3 + 12 $12.00/0

1994). The contractility of bovine trabecular meshwork can be modulated by an impressive number of drugs. However, the effect of trabecular meshwork contractility per se on outflow facility remains unclear. Isolated perfused eyes of primates and non-primates have been used for a long time to characterize the regulation of outflow facility (Leber, 1903; Ascher, 1942; Weekers, Watillon and Rudder, 1956; Macri, 1958: Langham, 1960; Grant, 1963; Brubaker, 1975; Neufeld et al., 1975; Kaufman and B~ir~iny, 1976; Hashimoto and Epstein, 1980; Epstein et al., 1982a, 1982b: Kaufman, 1984, 1986; Johnson and Tschumper, 1987; Erickson-Lamy, Rohen and Grant, 1988; Rosenquist et al., 1989; Erickson-Lamy et al., 1990; Johnson et al., 1990; Robinson and Kaufman, 1990: Erickson-Lamy, Rohen and Grant, 1991; Epstein, Hooshmand and Epstein, 1992; EricksonLamy and Nathanson, 1992 ; Liang et al., 1992). For anatomical reasons most of these models do not allow a complete dissociation of effects of ciliary muscle and trabecular meshwork on outflow regulation. Only in the bovine eye the ciliary muscle can easily be detached from the trabecular meshwork. An elegant study with detached ciliary muscle demonstrated that in the perfused anterior segment of the bovine eye trabecular meshwork cells maintain their morphological integrity at constant outflow resistance (Erickson-Lamy et al., 1988). In this model of the © 1995 Academic Press Limited

224

M. W I E D E R H O L T

perfused anterior segment, effects of substances which are used in glaucoma therapy were not tested. In studies with perfused eyes only adrenergic drugs (Kaufman, 1986; Robinson and Kaufman, 1990; Erickson-Lamy and Nathanson, 1992) were tested concerning their effect on outflow facility. The present paper indicates that substances which induce contraction in isolated trabecular meshwork strips reduce outflow of aqueous h u m o r by directly modifying the outflow pathway.

2. Materials and Methods

Perfusion of Anterior Segment Bovine eyes were obtained from a local slaughterhouse and transported on ice to the laboratory immediately after enucleation. The eyes were bisected cutting away vitreous and lens. In the anterior chamber segment the pectinate ligament was carefully detached and the iris and ciliary body including the ciliary muscle were gently peeled away. In this preparation only the trabecular (reticular) meshwork tissue remains on the corneoscleral segment (EricksonLamy et al., 1988; Flfigel et al., 1991). The anterior section was then rinsed with Ringer's solution and fixed to a Petri dish, using enbucrilate tissue glue

ET AL.

(histoacryl blue, Braun, Melsungen, Germany) on the cut edge. This procedure allows a tight fixation of the anterior chamber to the support without any distortion of the sclera. The aqueous veins were opened by a circular cut. The perfusion of the anterior section is shown schematically in Fig. 1. Two needles were placed through the cornea: near the limbus (perfusion) and the center of the cornea (application of substances). The perfusion reservoir, tubes, and needles were filled with prewarmed, filtered Ringer's solution. The reservoir was positioned 12 cm above the bottom of the anterior segment (pressure: 8"8 mmHg). The dish containing the perfused anterior segment was placed in a wet chamber with a heating system allowing a perfusion at 37°C. Preliminary experiments showed that only perfused eyes with an outflow rate of < 12 #1 min -1 resulted in stable outflow rates for at least 3 hr. In perfused segments with higher outflow rates at the beginning a ' w a s h o u t ' effect could be observed. Thus, only experiments with an initial outflow rate of < 12 #1 min -1 were accepted for testing the effect of drugs or pressure change on outflow facility. Usually, eight or nine out of ten preparations fulfilled these criteria and were used for experiments. To determine the perfusion rate of the anterior segment and thus the outflow rate, the perfusion reservoir was connected to a strain gauge (electric

Recorder Data storage

Application of substances

Wet chamber Perfusion <12 pl min-1 outflow

Outflow o0

o~

Perfusion solution

Temperature 37°C Fro. 1. Schematic diagram showing the perfusion system of the isolated anterior segment of the bovine eye.

R E G U L A T I O N OF B O V I N E O U T F L O W

balance) which recorded the decrease of perfusion reservoir weight. The volume was calculated from weight decrease. The perfusion reservoir was kept large enough to ensure that hydrostatic pressure was practically constant throughout the experiment. To avoid evaporation, the perfusion solution in the reservoir was .covered with silicone oil. After the experiments, the volume of the perfused compartment was measured and a m e a n value of 5 - 3 8 + 0 " 0 7 m l (n = 50) was obtained. By injecting methylene blue or lissamine green it could be shown that in this preparation the perfusion solution only leaves the segment via the aqueous veins. In preparations which were mechanically stable no leakage of stained Ringer's solution could be observed in the area of tissue glued to the Petri dish. Usually, an equilibration period of 1 hr was sufficient to obtain a stable outflow rate. The outflow was recorded for 1 hr (control period). At the end of the control period (time = 0 min) 100/zl of modified Ringer's solution with or without drugs was applied at time 0 and 1 hr later (experimental period). This volume of l O 0 / d ( < 2 % of the perfused compartment) was slowly injected over a period of 5 min and did not induce a pressure spike or alter the rate of outflow. Outflow was averaged for four consecutive periods of 10 min. For calculation of relative outflow the outflow at 0 min was normalized to 100%. Thus, the outflow in the control period was different from 100% (usually a few per cent > 100%). Outflow facility (C) was calculated as a ratio of the perfusion rate (/d min-') to the constant perfusion pressure (mmHg). Outflow resistance (R) was calculated as 1/C. In a n u m b e r of experiments the pressure in the perfused anterior segment was changed in small steps by changing the level of the perfusion reservoir (Figs 4. 5 and 8). For statistical analysis, the m e a n of four control values (each 10 min) were compared to the four last values of the first or second experimental period. Data are presented as mean_s.E.M. (paired Student's t-test).

225

109. Specimens of different areas of the four quadrants were analysed for light and electron microscopy.

Drugs The modified Ringer's solution of the following ionic concentrations was used (in mmol L'): NaCI, 151; KC1, 4: KH2P Q , 1; MgSO~, 0.9; CaC12.1'7: Hepes, 10. All solutions contained 5 m m o l l ' glucose. The pH was 7-4. Carbachol, atropin, pflocarpine, epinephrine and endothelin- 1 were supplied by Sigma (Deisenhofen, Germany). Metipranolol was a gift from Dr Mann P h a r m a (Berlin, Germany). 3. Results

Morphology In all of the ten perfused anterior segments the

Morphology For light and electron microscopy, ten anterior segments (after perfusion with control Ringer's for 1 hr and subsequent perfusion with carbachol 10 6 mol i 1 for 1 hr) were fixed in Ito's solution. The anterior segments were cut into four quadrants. Then the corneal portion anteriorly from the insertion line of the trabecular meshwork was removed. From the remaining sectors 1- to 2-mm-wide specimens were cut in a sagittal plane and embedded in Epon according to standard protocolls (F1/igel, T a m m and LtitjenDrecoll, 1991). Semi-thin sections (1 # m thick) were cut with an ultramicrotome and treated with Richardson's stain (Richardson, ]arret and Finke, 1960). Ultra-thin sections were stained with uranyl acetate and lead citrate and evaluated with a Zeiss EM

FIG. 2. Histological 1-/~m section through the outflow tissues (nasal quadrant, Richardson's stain, ×9). The trabecular meshwork (TM) is well preserved. Capillary loops (arrowhead) extend into the meshwork. At the posterior edge of the section a few individual muscle cells (arrow) are visible. Such muscle cells were only present in one or two of the four quadrants.

226

M. W l E D E R H O L T

ET AL.

FIG. 3. Electron micrographs of different regions of the outflow tissue. (A) Capillary loops (C) extending from the sclera into the filtering tissue ( x 440). (B) Myofibroblast-like cells (arrows) in the connective tissue strand between capillary loops and ciliary muscle ( x 4400). (C) Higher magnification of a myofibroblast-like presumably contractile cell ( x 12 000). The myofibrils are connected to the cell membrane by dense bands (arrowhead). The cell is incompletely surrounded by basements membrane material (arrow). (D) The cells in the trabecular meshwork are connected to each other by junctional complexes (arrowheads, x 4400). entire filtering tissue appeared well preserved. The trabecular/reticular meshwork consists of loosely arranged connective tissue strands covered by starlike tissue (Fig. 2). In the majority of specimens ciliary muscle cells could not be detected. Only in some specimens of the nasal and temporal quadrants where the filtering tissue is shorter than in the superior and inferior quadrants (Flfigel, T a m m and Lfitjen-Drecoll, 1991), some individual ciliary muscle cells were found (Fig. 2). As the electron microscopy showed, the ultrastructure of the filtering meshwork appeared well

preserved in all regions studied. The meshwork contains several capillary loops [Fig. 3(A)] which derive from larger vessels near the meshwork-sclera border. The endothelial cells of these loops contain typical giant vacuoles particularly in those areas where the vessels appear wider (not shown). The myofibroblast-like cells directly adjacent to the endothelium of the capillary loops [Fig. 3(B)] and within the large tissue strands between the outflow tissue and the cililary muscle [Fig. 3(B) and (C)] are well preserved. The myofibroblast-like cells show myofilaments, typical dense bands and an incomplete basal

227

R E G U L A T I O N OF B O V I N E O U T F L O W

TABLE [

Measured outflow and calculated outflow facility and resistance in perfused anterior segments of bovine eyes

Conditions

Outflow facility, C (/zl mmHg 1 min-')

Outflow resistance, R (mmHg min/zl 1)

7.66_+0.91 (7) 7"614- 1'02 7'08_+0"98

0"87 0"86 0'80

1.15 1'16 1'25

7"92 _+ 1.63 (7) 5"62 _+0"77*** 4"97___0"59***

0"90 0.64 0'56

1'11 2-56 2'79

6'92 _-4-1.00 (7) 6.76 4- 0.97 6-47 4- 0"96

0"79 0-74

1.27 1'30 1-35

8-28 4-O'91 (10) 6.88-+0"63"*

0"94 0'75

1"06 1'33

8"14___0-68 (9) 7"61 4-0"70*

0"93 0"86

1"O8 1"16

8"O2 + O"51 (7) 8"864- 0"81

0"91 1-00

1' 10 1"00

7"894- 0"63 (7) 7"11-+0"74"

0"90 0"81

1"11 1"23

6.89 + 0"67 (11) 5'28-+0"47"** 2"97 + 0'66***

0"78 0"60 0'34

1.28 1'67 2"94

6-23 -+ 0'38 (6) 4"33 4- 0"32***

0"71 0"49

1-41 2-04

Outflow (/d min -1)

Ringer's Control Exper. (0-1 hr) Exper. (1-2 hr) Carbachol (2 x 10 6 mol 1-1) Control Exper. (0-1 hr) Exper. (1-2 hr) Atropine (2 × 10 -~ mol 1 1) +Carbachol (2 x 10 8 mol 1-1) Control Exper. (0-1 hr) (Atropine) Exper. (1-2 hr) (Atrop. + Carb.) Pilocarpine (10 -5 mol 1-1) Control Exper. (0-1 hr) Epinephrine (10 -~ mol I 1) Control Exper. (0-1 hr) Epinephrine (10 -6 tool 1-1) Control Exper. (0-1 hr) Metipranolol (10 -4 mol I 1) +Epinephrine (10 6 mol 1-1) Control Exper. 0 - I hr) Endothelin (2 x 10 -9 mol 1-1) Control Exper. (0-1 hr) Exper. (1-2 hr) Endothelin (2 x 10 8 mol 1-1) Control Exper. (0-1 hr)

0"77

Anterior segments were perfused at constant pressure of 8.8 mmHg. At time zero and 1 hr 1O0/zl of Ringer's solution (with or without drugs) were injected into the chamber. Data are mean outflow rates 4-S.mM.Number of experiments are given in parenthesis. *P < 0.05 ; **P < 0-01 : •**P < 0,001 (vs. control).

l a m i n a [Fig. 3(C)]. J u n c t i o n a l complexes like gap j u n c t i o n s a n d m a c u l a r a d h a e r e n t e s are p r e s e n t [Fig. 3(D)]. The u l t r a s t r u c t u r e of t h e t r a b e c u l a r m e s h w o r k cells a p p e a r e d to be q u a n t i t a t i v e l y t h e s a m e as in the cells described p r e v i o u s l y (Flfigel, T a m m a n d LfitjenDrecoll, 1991).

Outflow Rate in Perfused Anterior Segments To s i m u l a t e the pressure drop across t r a b e c u l a r m e s h w o r k in vivo, a perfusion p r e s s u r e of 8"8 m m H g w a s used. This p r e s s u r e is in t h e s a m e r a n g e as h a s been used by Erickson-Lamy (Erickson-Lamy et al., 1988), a s s u m i n g a n i n t r a o c u l a r p r e s s u r e of 1 5 20mmHg a n d a n episcleral v e n o u s pressure of 10 m m H g (Booffel, 1 9 6 4 ; Erickson-Lamy et al., 1988). In both, Erickson-Lamy et al. ( 1 9 8 8 ) a n d o u r p r e p a r a tions, t h e a q u e o u s veins were opened, thus, the

pressure drop o c c u r r e d a l m o s t exclusively across t h e trabecular/reticular meshwork. Table I s u m m a r i z e s d a t a of e x p e r i m e n t s in w h i c h the relative outflow u n d e r c o n t r o l conditions (Ringer's solution) w a s m e a s u r e d over a period of 3 hr. Outflow w a s c o n s t a n t over t h a t period, a n d was n o t c h a n g e d by injection of 1 0 0 # 1 R i n g e r ' s solution into the a n t e r i o r s e g m e n t p r e p a r a t i o n t h r o u g h the needle for a p p l i c a t i o n of s u b s t a n c e s (Fig. 1) at time 0 m i n a n d 1 hr. The s a m e result w a s seen w h e n no a d d i t i o n a l v o l u m e w a s injected (data n o t shown). Table I further s u m m a r i z e s the absolute v a l u e s of outflow rate (/zl m i n 1) a n d t h e c a l c u l a t e d outflow facility a n d resistance for all s u b s t a n c e s tested in this study. W h e n c a l c u l a t i n g the outflow rate of all control periods, a m e a n v a l u e of 7'55___0.23/zl m i n ~ (81 experiments, n i n e different c o n t r o l groups) w a s obtained. The m e a n outflow facility w a s 0 " 8 6 + 0 . 0 3 / z l m m H g -~ m i n -~

228

M. W I E D E R H O L T ET AL.

250

-

(A) I 4.4 mmHg

8.8 mmHg

t

4-4 mmHg

]

200 o

o

~ 150

100

50

I 0 (B)

~

,

I 30

[ 8-8 mmHg

~

~

r 60

~

~

I 90

~

4-4 mmHg

30

I 120

8.8 mmHg

,

0

,

60 Time (rain)

,

,

90

]

,

120

Fro. 4. Effect of stepwise change of perfusion pressure on relative outflow rate. The outflow was normalized to 100 % at time zero. In (A) the sequence was down-up, in (B) up-down. Numbers are average_+ S.E.M. for eight perfused eyes. 50 40

"~'~ 30

.~/~"-"-

10 I

0

10

20 30 40 Perfusion pressure (mmHg)

50

Fro. 5. Relation between mean relative outflow rates and perfusion pressure. Each value represents the mean of eight different eyes±s.E.M. The pressure sequence in all experiments was as follows: 10, 5, 15, 20, 25, 30, 35, 40, 45, 50, 10 mmHg. (range 0"71-0"94) and the resistance m m H g min,ul -~ (range 1-06-1"41).

1.17_+0.04

Effect of Pressure on Outflow To test the relation between flow rate and perfusate into the anterior chamber, the pressure in the anterior c h a m b e r was changed by varying the level of the reservoir. Figure 4 demonstrates that the m e a n

outflow was reversibly dependent on the perfusion pressure. The sequence of perfusion pressure c h a n g e [ d o w n - u p sequence, Fig. 4(A); u p - d o w n sequence, Fig. 4(B)] did not influence the absolute values and the reversability of the outflow. Experiments similar to those s h o w n in Fig. 4 were performed for the perfusion pressure sequence 8-8, 13.2, 8-8 m m H g (data not shown). Figure 5 is a graphic representation of flow versus pressure of experiments with pressure sequence

R E G U L A T I O N OF B O V I N E O U T F L O W

229

120 Atropine 110

; 100

Atropine 2 x 104 tool 1-1

-

~ 9080_ ~°

+ Carbachol 2 x 104 mol 1-1

~NN~***~"~"~

_.

70 60

Carbaehol 2 x 104 mol I-] i 0

~

,

I 30

I ~

~

I , 60 Time (min)

_

~

~

~

~--~

,

I 90

J

,

I 120

Fro. 6. Typical plot of experimentally measured outflow during perfusion at constant pressure of 8"8 mmHg. After an equilibration period of 1 hr the outflow was normalized to 100% at time zero. In all experiments 100/~1 Ringer's solution with or without drugs was applied at 0 min and 60 min. In the experimental periods carbachol or atropine and carbachol were added. Each value represents the mean and S.E.M. of seven experiments. Carbachol reduced the outflow significantly (*P < 0"05; ***P < 0-001) as compared to control conditions (data not shown) or pretreatment with atropine.

30 25

***

***

t

o

20

<9 N 15 ~

10

N

5

10 -10

10-9

104

10-7

104

10-5

10-4

[Carbachol] (mol 1-]) FIG. 7. Dose-response curve of the carbachoMnduced inhibition of the relative outflow. Each value represents the mean_s.E.M, of seven experiments. ECso was 3 x 10 -8 mol I 1. **p < 0.01 ; ***P < 0.001 (vs. no inhibition).

as follows: 10, 5, 15, 20, 25, 30, 35, 40, 45, 50, 10 m m H g . It c a n be seen t h a t outflow rate w a s a n a l m o s t linear function of p r e s s u r e in the r a n g e of 5 to 25 m m H g . A t h i g h e r perfusion pressure the i n c r e a s e of outflow r a t e b e c a m e smaller, i n d i c a t i n g a n i n c r e a s e of outflow resistance. This o b s e r v a t i o n h a s b e e n first described for the perfused h u m a n eye (Grant, 1963).

Carbachol and Pilocarpine on Outflow As s h o w n in Fig. 6 a n d Table I c a r b a c h o l 2 x 10 -6 m o l 1 1 a d m i n i s t e r e d at time O m i n a n d 1 hr, significantly r e d u c e d outflow o n l y 10 m i n after application, r e a c h i n g a 2 5 - 3 0 % inhibition approxim a t e l y 1 h r later. P r e t r e a t m e n t w i t h a t r o p i n e c o m pletely blocked the effect of c a r b a c h o l o n outflow.

A t r o p i n e per se did n o t influence t h e outflow r a t e as c a n be derived from the a b s o l u t e flow rates s h o w n in Table I (Ringer's solution versus atropine). Figure 7 gives a sigmoidal d o s e - r e s p o n s e c u r v e with c a r b a c h o l c o n c e n t r a t i o n s r a n g i n g from 2 x 10 -l° to 10 -4 m o l 1-1. A m a x i m a l inhibition of outflow resulted at c o n c e n t r a t i o n s of 10 6 tool 1-1 a n d h i g h e r with a n ECso of 3 x 1 0 - S m o l l 1. In a series of e x p e r i m e n t s the effect of c a r b a c h o l o n outflow r a t e w a s tested at different perfusion pressures (Fig. 8). The s e q u e n c e of perfusion pressure c h a n g e w a s as follows: 4'4, 7.4, 10.3, 13.2, 4 . 4 m m H g . A s s u m i n g a linear i n c r e a s e of outflow with i n c r e a s i n g perfusion pressure ( 4 . 4 - 1 3 - 2 m m H g ) , a m e a n outflow resistance of 0 . 8 5 4 - 0 . 1 5 m m H g m i n # l 1 w a s calculated for c o n t r o l e x p e r i m e n t s . C a r b a c h o l significantly

230

M. W I E D E R H O L T

ET AL.

Perfusion pressure (mmHg)

4.4

~

300

~ 200

100 0

30

60

90 Time (min)

120

150

170

FIG. 8. Determination of the effect of carbachol (10 .8 tool 1-1) on outflow rate and outflow resistance at four different perfusion pressures (4-4, 7.4, 10.3 and 13-2 mmHg). Mean_+s.E.M. of nine experiments. ©, control; O, carbachol.

n.s.

P<0.05

r

]

Control

Epinephrine 10~ mol 1-1

i

P<0.05 i

r

]

110

100

~ 9o ~" 8o 70

Metipranolol Metipranolol 10-~ mol 1-1 + Epinephrine 10~ mol 1-1

Control

Epinephrine 10-5 mol 1-1

FIG. 9. Summary of the effect of the epinephrine on relative outflow. Inhibition of the epinephrine (10 -Gmol l-~)-induced effect by the fl-adrenergic antagonist metipranolol. Mean values + S.E.M. Number of experiments is given in parenthesis.

increased outflow resistance to 1-34___0.22 m m H g min ,a1-1 (n = 9, P < 0.001). To correlate morphology and function, in addition to the experiments mentioned above, ten experiments were performed in which the specimens were prepared for morphology after perfusion with carbachol (see Methods, and Figs 2 and 3). In five experiments with a few remaining ciliary muscle cells (demonstrable only in some specimens of the nasal and temporal quadrants, Fig. 2) from the transitional region between reticular meshwork and ciliary muscle (F1/igel, T a m m and L/itjen-DrecolL 1991), carbachol reduced the outflow from 101.3_+0.9% (before carbachol) to 77.3 -+2.4% (60 min after carbachol, P < 0"001). The

effect of carbachol was statistically significant (P < 0.05) already after 10 min of application of the drug. In five experiments the transitional region was totally destroyed by deep circular cuts with a razor blade. In these experiments, the effect of carbachol was identical, reducing the relative outflow from 1 0 2 . 4 + 2 . 4 % to 8 6 . 9 + 1 ' 3 % . Again, the effect of carbachol was statistically significant 10 min after application of the drug. Pilocarpine was tested in a dose which submaximally contracted isolated trabecular meshwork strips (Wiederholt et al., 1993). After 1 hr of perfusion, pilocarpine 10-Smol 1 1 significantly (P < 0.01) reduced the outflow rate from 8'3 to 6'9 #1 min -1

REGULATION OF BOVINE OUTFLOW

231

was seen within the second hour after application. Endothelin 2 x 10 -8 mol 1-1 was even more effective in changing outflow rate, outflow facility and resistance (Table I). The doses used are comparable to those which lead to maximal contraction of isolated bovine trabecular meshwork strips (Lepple-Wienhues et al., 1991b).

10

i

4. Discussion o

o

~I

*** i

1 hr

2 hr

(11)

(11)

I Control

Endothelin 2 × 10-9 tool 1-1

Fro. lO. Inhibition of relative outflow by endothelin 2 x l O 9moll 1 at O, 1 and 2hr. Mean+s.E.M. (n= 11); ***P < 0'001. (Table I). This inhibition (15 %) of outflow rate is less than the effect of carbachol when comparing equimolar concentrations.

Epinephrine on Outflow It is well known that at high concentrations, epinephrine activates both c¢- and fl-adrenergic receptors. In a concentration of 10 -5 tool 1-' epinephrine significantly reduced outflow by 15 % (P < 0-05) as measured 1 hr after application (Fig. 9). On the other hand, a lower concentration (10 -6 mol 1-i) of epinephrine increased the outflow rate in seven out of ten single experiments. This increase in outflow rate was not significant statistically (Table I). However, pretreatment with metipranolol 10 4 tool 1 1 completely blocked the effect of epinephrine 10 -6 tool 1 1 on outflow (P < 0.05) and induced a reduction of outflow rate to the same level as with epinephrine 10 -5 mol 1-1 (Fig. 9).

Endothelin on Outflow In Fig. 10 a s u m m a r y of experiments is given in which endothelin 2 x 10 -9 mol 1-1 induced a significant reduction of relative outflow which could already be observed within the first hour after application. A further significant (P < 0"001) reduction of outflow

In this study, we have found that substances which contract isolated bovine meshwork strips (LeppleWienhues et al., 1991a, 1991b; Wiederholt et al., 1993) are changing the outflow pathway in such a way that a reduction of outflow rate and an increase of outflow resistance occurs. Since in the bovine eye the ciliary muscle is rudimentary and posteriorly located, the ciliary muscle can easily be separated from the trabecular meshwork, which more closely resembles a reticular meshwork (Flfigel et al., 1991). Recently, histochemical (Gierson et al., 1986: DeKater et al., 1990: F1/igel et al., 1991) and electrophysiological (Coroneo et al., 1991) evidence has been presented for a direct contribution of contractile elements of the bovine meshwork in regulation of aqueous h u m o r outflow. However, the functional importance of these contractile elements in relation to outflow has not been demonstrated. Outflow measurements have been performed in isolated h u m a n eyes and anterior ocular segments (Leber, 1903; Ascher, 1942; Grant, 1963; Brubaker, 1975; Johnson and Tschumper, 1987; Erickson-Lamy et al., 1990; Johnson et al., 1990; Erickson-Lamy et al., 1991; Liang et al., 1992) as well as in various preparations of eyes from different m a m m a l i a n species (Kaufman and B~ir~iny, 1976; Epstein et al., 1982; Kaufman, 1984; Erickson-Lamy et al., 1988 ; Johnson et al., 1990) including the bovine eye (Weekers et al., 1956: Hashimoto and Epstein, 1980: Erickson-Lamy et al., 1988). Several drugs which are of some interest for antiglaucoma therapy have been tested. However, the exact location of the action of the drugs could not be determined because of the interaction of the ciliary muscle with the trabecular meshwork. In primate eyes, Kaufman et al. (Kaufman and B~ir~iny, 1976) made an attempt to dissociate the effect of pilocarpine on ciliary muscle and trabecular meshwork by disinsertion of the ciliary muscle. In this study, the lack of cholinergic response of the trabecular meshwork could be due to the increased outflow resistance induced by the disinsertion, making it difficult to observe a further increase of resistance by cholinergic treatment. However, the authors interpreted their data as evidence for a disappearance of cholinergic response because of the lack of the longitudinal ciliary muscle effect. Due to the anatomical situation, the bovine eye provides a good model to test the effect of drugs on the trabecular outflow pathway directly. In our anterior segment perfusion preparation, outflow

232

M. W I E D E R H O L T

ET AL.

TABLE II

Outflow resistance as a function of intraocular perfusion pressure in calf eyes

Method Enuc|eated eye Enucleated eye Aqueous outflow pathway Aqueous outflow pathway

Perfusion pressure (mmHg)

Outflow resistance (mmHg min/d 1)

15 45 15 45 15 45 15 45

0"55 0"80 0.63 1"10 0.69 1'30 0'89 _+0'06* 1"34_+0"12"

Source Fig. 1, p. 228 Fig. 4, p. 1486 Fig. 1, Table I, p. 802 Fig. 5

Reference Weekers, Watillon and De Rudder (1956) Hashimoto and Epstein (1980) Erikson-Lamy, Rohen and Grant (1988) Present study

* M e a n + s . E . M . (n = 8; P < 0 . 0 1 ) .

measurements could be performed which were stable for several hours. In an earlier study, it had been shown that in such an anterior segment perfusion, the morphological integrity was maintained (EricksonLamy et al., 1988). It was found that disinsertion of the ciliary muscle does not change the outflow facility compared to the intact anterior segment (EricksonLamy et al., 1988). We are also presenting evidence that the meshwork cells of the perfused anterior segment of the bovine eye quantitatively are the same as in the intact eye. Furthermore, the morphology shows that in the perfused anterior segment trabecular/reticular meshwork cells are dominating, with only a few ciliary muscle cells in some areas of the nasal and temporal quadrants of the transitional region. These few remaining ciliary muscle cells are functionally negligible. Carbachol significantly reduced outflow rate in preparations where the transitional region was totally destroyed and in preparations where a very small fraction of this region remained. In our preparation with a constant perfusion pressure of 8.8 mmHg, we did not observe a ' w a s h o u t ' effect with time of perfusion as has been found in the perfused eyes of various species other t h a n the h u m a n eye (Erickson-Lamy et al., 1990). A progressive increase in outflow facility with time of perfusion is called ' w a s h o u t ' . From inspection of Figs 4, 6, and 8 it can be seen that in our experiments the outflow facility did not increase but had a tendency to decrease. This effect, however, was not significant statistically. Erickson-Lamy et al. (1988) also reported that ' w a s h o u t ' is minimal or non-existent in calf eyes at a perfusion pressure of 6 m m H g (p. 801, reference 14). On the other hand, we observed a ' w a s h o u t ' effect when tissues were perfused at pressures above 15 m m H g for more t h a n 2 hr. A perfusion pressure of 15 m m H g was used in experiments on neonatal calf eyes showing a ' w a s h o u t ' effect (Erickson-Lamy et al., 1990). Since the normal pressure drop across the trabecular meshwork is m u c h less than 15 m m H g (see Results), a high perfusion pressure could be the reason

for a time-dependent damage of the outflow segment leading to a ' w a s h o u t ' of essential components within the outflow pathway. There is accumulating evidence that an increase in intraocular pressure per se m a y increase the outflow resistance. Most evidence for this hypothesis has been derived from experiments with perfused eyes of various species (for review, Brubaker, 1975). In a most careful study, Brubaker (1975) presented convincing evidence that in the enucleated perfused h u m a n eye, outflow resistance increases directly with intraocular pressure, i.e. higher intraocular pressure induced higher resistance values. There is very little information on the mechanism of this resistance change. Table II summarizes data obtained in perfused calf eyes. Our data on resistance are in good agreement with published data. It is of special interest that in the study of Erickson-Lamy, Rohen and Grant (Erickson-Lamy et al., ] 988), and in our own study where only anterior segments without ciliary muscle were perfused, outflow resistance increased at higher perfusion pressure. Thus it is most likely that increase of intraocular pressure directly increases resistance of the aqueous outflow pathway. Although the perfnsed anterior segment of the bovine eye could be a good model (Epstein et al., 1982 ; Erickson-Lamy et al., 1988; Johnson et al., 1990) to test the effect of drugs which are aimed to modulate trabecular outflow pathway, an extensive p h a r m a cological study, especially in a model with detached ciliary muscle, is missing. We have shown that cholinergic and adrenergic agents and endothelin induce contractions in isolated bovine trabecular meshwork strips (Lepple-Wienhues et al., 1991a, 1991b, 1992b: Wiederholt et al., 1993) and depolarize the m e m b r a n e voltage of cultured bovine (and h u m a n ) trabecular meshwork cells (Coroneo et al., 1991; Lepple-Wienhues et al., 1992a, 1994; Wiederholt et al., 1993). These cultured cells exhibited electrophysiological properties which are typical for contractile smooth muscle-like cells. The present data show that carbachol dose-dependently reduced the

R E G U L A T I O N OF B O V I N E O U T F L O W

bovine outflow and thus decreased outflow facility and increased resistance (Fig. 7, Table I). The ECso of carbachol on outflow rate was in the same range as the effect of carbachol on isolated trabecular meshwork strips. Furthermore, comparing the effect of 10 5 mol 1 1 pilocarpine with the equimolar effect of carbachol, the. difference in the effectiveness of both drugs on outflow rate resembled those on contractility of isolated meshwork strips. The effect of epinephrine to increase outflow facility and thus lower intraocular pressure has been explained by direct actions of epinephrine on the t r a b e c u l a r meshwork (Kaufman, 1984, 1986: Alvarado et al., 1990; Erickson-Lamy and Nathanson, ] 992). Owing mainly to species differences it remained an open question whether the effects on outflow facility are mediated by ~- or fl-adrenergic receptors. The outflow pathway in the primate eye seems to be mainly regulated by fl-adrenergic mechanisms (Kaufman, 1984; Alvarado et al., 1990). A direct effect of epinephrine on the morphology of meshwork cells has been demonstrated (Tripathi and Tripathi, 1984). Since epinephrine is a non-specific ~- and fladrenergic agonist, an interpretation of the mech"anisms of action are complicated. In isolated strips of the bovine trabecular meshwork high concentrations of epinephrine induced contractions (Wiederholt et al., 1993). A further contraction was induced w h e n the flcomponent of the drug was specifically blocked by metipranolol. On the other hand, the contraction induced by a-adrenergic drugs could be selectively inhibited by antagonists, indicating effective functional a- and fl-adrenergic receptors in the bovine outflow pathway. This concept is supported by our outflow measurements (Table I, Fig. 9) showing a reduction of the outflow rate with a high dose of epinephrine and a small increase of the outflow with a lower dose of epinephrine (10 6 tool 1-1). In the perfused h u m a n eye, the effect of 10 _6 mol 1-1 epinephrine on increase of outflow was more pronounced (Erickson-Lamy and Nathanson, ]992). Thus, it is important that in our experiments the effect of epinephrine was fully blocked by the fl-antagonist metipranolol. The relative activity of a- and fl-adrenergic stimulation determines whether epinephrine induces contraction or relaxation of the trabecular meshwork and thus increases or decreases the outflow facility. Endothelin has been shown to increase outflow in the perfused monkey eye, probably by a direct effect on the ciliary muscle (Erickson-Lamy et al., 1991). In the perfused anterior segment of the bovine eye without ciliary muscle, endothelin dose-dependently decreased outflow rate and increased outflow resistance (Table I). Again, these data are compatible with the contraction of isolated meshwork strips induced by endothelin (Lepple-Wienhues et al., 199 l b). Since substances like carbachol, pilocarpine, endothelin and epinephrine (high dose) contract isolated ciliary muscle and trabecular meshwork strips, it can

233

be concluded that the direct effect of these drugs on the trabecular meshwork cells leads to a decrease of the outflow rate in the bovine eye, thereby increasing the outflow resistance. Since it has been shown that cholinergic mimetics (Kaufman, 1984) and endothelin (Erickson-Lamy et al., 1991) decrease intraocular pressure and increase overall outflow facility, the direct effect of these substances on contractility of the bovine trabecular meshwork cells is functionally antagonistic to the direct effect on ciliary muscle. Thus intertrabecular spaces could be narrowed by contracting trabecular fibres and widened by ciliary muscle traction. An alternative possibility might exist: in the presence of both tissues, contraction of the trabecular meshwork m a y increase the rigidity of this tissue allowing the ciliary muscle contraction to be more effective in altering the geometry of the trabecular meshwork. Although contraction of the ciliary muscle dominates the overall effect on outflow facility in the h u m a n (and most likely bovine) eye, the concept of antagonism between ciliary muscle and trabecular meshwork has to be considered in the interpretation of mechanism of action of currently used antiglaucoma drugs and in the search for new effective drugs. Although the application of the bovine model to the h u m a n eye has to be tested, there are m a n y similarities between isolated ciliary muscle and trabecular meshwork systems (including cultured cells) of the bovine and the h u m a n eye (Wiederholt et al., 1993). The perfused aqueous outflow pathway of the bovine eye is a useful tool to study the complex physiology and pharmacology of the chamber angle.

Acknowledgements Supported by the Deutsche Forschungsgemeinschaft (Wi 328/11), Maria Sonnenfeld-Gedfichtnis-Stiftung (stipends for S.B. and F.S.) and Dr Mann Pharma (Berlin).

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